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Effect of high pressure treatment on proteolytic system in meat
Hayashi and C. Balny (Editors), High Pressure Bioscience and Biotechnology 9 1996 Elsevier Science B.V. All rights reserved.
R.
327
Effect of high pressure treatment on proteolytic system in meat Noriyuki Homma, Yoshihide Ikeuchi and Atsushi Suzuki Department of Applied Biological Chemistry, Faculty of Agriculture, University of Niigata, Niigata-shi, Niigata 950-21, Japan Abstract High hydrostatic pressurization increased the total activities of lysosomal cathepsins and calpains in the muscle. The pressure-induced increase in the amount of cathepsin activities was due to the release of enzymes from lysosomes. On the other hand, the increase of calpain activities was caused by the increase of Ca '+ release from sarcoplasmic reticulum, and inactivation of calpastatin. These increses of activities may result in tenderization of meat.
1. INTRODUCTION Generally, postmortem tendefization of meat during storage is caused by the proteolytic enzymes. Mainly, two enzymic systems(cathepsins and calpains) have been implicated. Catheptic enzymes are released from lysosomes, due to the rupture of lysosomal membranes and may promote ageing by proteolysis of myosin, actin, cractinin, troponin T and troponin I. [1, 2] Calpain removes the Z-disc from myofibrils in the presence of Ca '+ and causes many changes in myofibrillar structure which could be related to increased meat tenderness. [3, 4] High hydrostatic pressurization is one of the new technologies for tenderizing meat or accelerating meat conditioning. The changes in the ultrastructure of the pressurized muscle have been reported by many workers. [5-7] It seemed that the pressureinduced changes in the muscle structure were derived from not only the physical force but also the increase of the proteolytic activity of enzymes in the muscle. Therefore we measured the changes of activity of lysosomal enzymes and calpain system induced by the pressurization. 2. LYSOSOMAL ENZYMES Changes of lysosomal enzyme activity extracted from the pressurized muscle are shown in Fig.1. Activity of acid phosphatase increased with increasing pressure applied to the muscle up to 500 MPa. Activity of cathepsin B, D and L increased up to 400 MPa, then tended to decrease at 500 MPa. Aminopeptidase B decreased with the increasing pressure.
328
Measurements of enzymic activity in the pressurized crude extract, to investigate the pressure effect on the enzymes themselves, showed that all lysosomal enzymes lost their enzymic activity as applied pressure increased. (Fig.2) Acid
Phosphat
s
)
Acid P h o ~ p ~ a t a s e Cathepsi Cathepsin L
Cathepsin Cathepsin
B 50.
_l
L
Cathepsi; Aminopep
Cathepsin Cathepsin (Aminopeptidas Aminopeptidase C ~
1~
2~
3~
4~
i
ase
B
Cathepsin ~ (Aminopepti ase
B)
B
ConUol
5~
100
200
300
400
500
Pressure ( MPa )
Press~e(MPa)
Fig.2.Changes of the enzymic ability in the pressurized crude extract.
Fig.l.Changes of enzyme activity extracted from pressurized muscle.
When the pressurized extracts were subjected to the gel-filtration chromatography (Fig. 3), a decrease in the activity of aminopeptidase B and an increase in the activities of cathepsin B and L and acid phosphatase were observed. It seems that the decrease in activity of the enzymes eluted early from the column (aminopeptidase B) is due to the decrease of the amount of the eluted protein,whereas the increase of activity of the enzymes eluted late (cathepsin B, L and acid phosphatase) is due to the increase of the amount of the protein eluted. Cathepsin 3o eC o 03
Cathepsin
B
on
r
,o
@Control
L
Aminopeptidase 41
~
a
B
eControl
Acid
0.~
30OHPa
20
Phosphatase
eControl 03
a
v o~
i,o o
io
is
2o
2s
Frlclmn numOeq
30
io
Is Fractk~
2o
zs
3o
number
Io
is Fr
20
2S
numbl~
3O
1~
15
2O
2S
3O
Fr~llon number
Fig.3.Gel filtration chromatographs and enzymes activities of crude extract from control and pressurized muscle.
to
15
20
zs
30
Friction mm~e*
329 These restllts showed that the substances of large molecular weight tended to be degraded by the pressure treatment, and the amount of activity was influenced by amount of eluted protein solution in each fraction. Therefore, it was concluted that the pressure-induced increase in the amount of cathepsin activities was due to the release of enzymes from lysosomes. This increse might result in tenderization of meat.
3. CALPAIN SYSTEMS The changes of /.z-, m-calpain and calpastatin levels in the pressurized rabbit muscle are shown in Fig.4 and Table 1. Calpains and calpastatin (specific inhibitor) lost their activity with increasing pressure, but the degree of loss was different for each. Calpains resisted changes in pressurization at 200MPa and were inactivated over 200 MPa. Inactivation of calpastatin at 100MPa was faster than that of calpains. Cont 6.0
9
Inhibit
co
o r 7"
i m t
,'"
/
i
200MPa
"~
.
.
60
70
;"~
~_
80
90
100
_~.
110
,o
......
120
Inhibitor
6"O-'le
. o . 5 .~
"~
~
~o
I'
.
,- I 0
m
~
130
140
/"
//
-o.1
.
o.
"1.5
;T
"
~,
-0.3
o
o
"
150
,
,
~
60
70
80
100
110
FRACTION NUMBER
300MPa
~
/
~
-5o
-
- o . 5 ~_
il~
100MPa
-0.2 -,.o ~
o.,
o
-0 90
120
130
140
150
"'"" ,.o 9-
.
,-"
-o.1 -o.s~i
LO 60
70
80
90
100
.~
~
FRACTION NUMBER
~.
[
0.2 -1.0 ~IE
" ol.
-
i
0.3 -I.5
100
110
FRACTION NUMBER
120
130
140
1500
-0
~
,-"" ~
o-L
, 60
, 70
a--80
~ 90
~ e ~ - - * - - " r 100 110 120 130
~-0.1
-o.5
o '-o 140
150
FRACTION NUMBER
Fig.4.Elution profile of DEAE-Sephacel of the extract from muscle. Comparatively, Zz-calpain appeared to decrease more than m-calpain at 200MPa pressurization. It was considered as follows. At 200MPa, which had no effect on m-calpain,the level of /_z-calpain decreased rapidly, because in the pressurized muscle, Ca 2, concentration was probably increased so far as /_z-calpain was activated.
330 In short, at 200MPa, r activated by an increase in Ca ~§ may have decreased thep level of Zz-calpain by autolysis. Suzuki et al. [8] presented direct evidence for the pressure-induced Ca '.+ release from sarcoplasmic reticulum from electron micrographs of the pyroanthimonate-fixed fiber bundles prepared from pressurized muscles. Emori [9] reported that the primary structures of the /.z- and m-calpain large subunits were similar to one another. On the basis of these reports, it was difficult to accept that the two calpains had a different ability to withstand pressurization. From the results,it was concluded that calpain levels remained in muscle pressurized up to 200 MPa, whereas calpastatin level in the muscle was decreased and Ca ~+ concentration increased by the pressurization. Therefore,the total activities of calpains in the pressurized muscle were increased, and could result in tenderization of meat. P?essure Applied
=-Calpain
Control
100
m-Calpain 100
100
IOOMPa
55.9
96.4
39.6
200MPa
5.4
99.5
14.5
300MPa
1.6
8.2
Values are calculated
the on
in the
vitro basis
activities of the
Table 1 Relative levels of calpain and calpastatin after pressurization
Calpastatin
4.3 of each chromatographs.
peak,
4. CONCLUSION From the results, it was concluded that the pressure-induced increase in the amount of protease activity was due to the release of the enzymes from lysosomes. And total activities of calpains in the pressurized muscle were increased by the pressure treatment. These increase of activity may result in tenderization of meat. 5. REFERENCES
1 W. N. Schwartz and J. W. C. Bird, Biochem. J., 167 (1977) 811. 2 A. Okitani, U. Matsukura, H. Kato and M. Fujimaki, J. Biochem., 87 (1980) 1133. 3 D. E. Goll, M. H. Stromer, D. G. Olson, W. R. Dayton, A. Suzuld and R. M. Robson, Proc.of the Meat Industry Res. Conf., Am. Meat Inst. Found. Arlington, Virginia, 1974 4 A. Suzuld and D. E. Goll, Agric. Biol. Chem., 38 (1974) 2167. 5 J. J. Macfarlane, Developments in Meat Science-3(R. A. Lawrie, ed. ), Elsevier Applied Science Publishers, Barking, Essex, 1985 6 E. A. Elgasim and W. H. Kennick, Food Microstructure, 1 (1982) 75. 7 A. Suzuki, K. Kim, N. Homma, Y. Ikeuchi and M. Saito, High Pressure and Biotechnology. (C.Balny et al., eds.), Colloques INSERM/John Libbey Eurotext, Montrouge, 1992 8 Y. Emori, H. Kawasaki, S. Imajoh, S. Kawashima and K. Suzuki, J. Biol. Chem., 261 (1986) 9472. 9 A. Suzuki, A. Okamoto, Y. Ikeuchi and M. Saito, Biosci. Biotech. Biochem., 57 (1993) 862.
R.
327
Effect of high pressure treatment on proteolytic system in meat Noriyuki Homma, Yoshihide Ikeuchi and Atsushi Suzuki Department of Applied Biological Chemistry, Faculty of Agriculture, University of Niigata, Niigata-shi, Niigata 950-21, Japan Abstract High hydrostatic pressurization increased the total activities of lysosomal cathepsins and calpains in the muscle. The pressure-induced increase in the amount of cathepsin activities was due to the release of enzymes from lysosomes. On the other hand, the increase of calpain activities was caused by the increase of Ca '+ release from sarcoplasmic reticulum, and inactivation of calpastatin. These increses of activities may result in tenderization of meat.
1. INTRODUCTION Generally, postmortem tendefization of meat during storage is caused by the proteolytic enzymes. Mainly, two enzymic systems(cathepsins and calpains) have been implicated. Catheptic enzymes are released from lysosomes, due to the rupture of lysosomal membranes and may promote ageing by proteolysis of myosin, actin, cractinin, troponin T and troponin I. [1, 2] Calpain removes the Z-disc from myofibrils in the presence of Ca '+ and causes many changes in myofibrillar structure which could be related to increased meat tenderness. [3, 4] High hydrostatic pressurization is one of the new technologies for tenderizing meat or accelerating meat conditioning. The changes in the ultrastructure of the pressurized muscle have been reported by many workers. [5-7] It seemed that the pressureinduced changes in the muscle structure were derived from not only the physical force but also the increase of the proteolytic activity of enzymes in the muscle. Therefore we measured the changes of activity of lysosomal enzymes and calpain system induced by the pressurization. 2. LYSOSOMAL ENZYMES Changes of lysosomal enzyme activity extracted from the pressurized muscle are shown in Fig.1. Activity of acid phosphatase increased with increasing pressure applied to the muscle up to 500 MPa. Activity of cathepsin B, D and L increased up to 400 MPa, then tended to decrease at 500 MPa. Aminopeptidase B decreased with the increasing pressure.
328
Measurements of enzymic activity in the pressurized crude extract, to investigate the pressure effect on the enzymes themselves, showed that all lysosomal enzymes lost their enzymic activity as applied pressure increased. (Fig.2) Acid
Phosphat
s
)
Acid P h o ~ p ~ a t a s e Cathepsi Cathepsin L
Cathepsin Cathepsin
B 50.
_l
L
Cathepsi; Aminopep
Cathepsin Cathepsin (Aminopeptidas Aminopeptidase C ~
1~
2~
3~
4~
i
ase
B
Cathepsin ~ (Aminopepti ase
B)
B
ConUol
5~
100
200
300
400
500
Pressure ( MPa )
Press~e(MPa)
Fig.2.Changes of the enzymic ability in the pressurized crude extract.
Fig.l.Changes of enzyme activity extracted from pressurized muscle.
When the pressurized extracts were subjected to the gel-filtration chromatography (Fig. 3), a decrease in the activity of aminopeptidase B and an increase in the activities of cathepsin B and L and acid phosphatase were observed. It seems that the decrease in activity of the enzymes eluted early from the column (aminopeptidase B) is due to the decrease of the amount of the eluted protein,whereas the increase of activity of the enzymes eluted late (cathepsin B, L and acid phosphatase) is due to the increase of the amount of the protein eluted. Cathepsin 3o eC o 03
Cathepsin
B
on
r
,o
@Control
L
Aminopeptidase 41
~
a
B
eControl
Acid
0.~
30OHPa
20
Phosphatase
eControl 03
a
v o~
i,o o
io
is
2o
2s
Frlclmn numOeq
30
io
Is Fractk~
2o
zs
3o
number
Io
is Fr
20
2S
numbl~
3O
1~
15
2O
2S
3O
Fr~llon number
Fig.3.Gel filtration chromatographs and enzymes activities of crude extract from control and pressurized muscle.
to
15
20
zs
30
Friction mm~e*
329 These restllts showed that the substances of large molecular weight tended to be degraded by the pressure treatment, and the amount of activity was influenced by amount of eluted protein solution in each fraction. Therefore, it was concluted that the pressure-induced increase in the amount of cathepsin activities was due to the release of enzymes from lysosomes. This increse might result in tenderization of meat.
3. CALPAIN SYSTEMS The changes of /.z-, m-calpain and calpastatin levels in the pressurized rabbit muscle are shown in Fig.4 and Table 1. Calpains and calpastatin (specific inhibitor) lost their activity with increasing pressure, but the degree of loss was different for each. Calpains resisted changes in pressurization at 200MPa and were inactivated over 200 MPa. Inactivation of calpastatin at 100MPa was faster than that of calpains. Cont 6.0
9
Inhibit
co
o r 7"
i m t
,'"
/
i
200MPa
"~
.
.
60
70
;"~
~_
80
90
100
_~.
110
,o
......
120
Inhibitor
6"O-'le
. o . 5 .~
"~
~
~o
I'
.
,- I 0
m
~
130
140
/"
//
-o.1
.
o.
"1.5
;T
"
~,
-0.3
o
o
"
150
,
,
~
60
70
80
100
110
FRACTION NUMBER
300MPa
~
/
~
-5o
-
- o . 5 ~_
il~
100MPa
-0.2 -,.o ~
o.,
o
-0 90
120
130
140
150
"'"" ,.o 9-
.
,-"
-o.1 -o.s~i
LO 60
70
80
90
100
.~
~
FRACTION NUMBER
~.
[
0.2 -1.0 ~IE
" ol.
-
i
0.3 -I.5
100
110
FRACTION NUMBER
120
130
140
1500
-0
~
,-"" ~
o-L
, 60
, 70
a--80
~ 90
~ e ~ - - * - - " r 100 110 120 130
~-0.1
-o.5
o '-o 140
150
FRACTION NUMBER
Fig.4.Elution profile of DEAE-Sephacel of the extract from muscle. Comparatively, Zz-calpain appeared to decrease more than m-calpain at 200MPa pressurization. It was considered as follows. At 200MPa, which had no effect on m-calpain,the level of /_z-calpain decreased rapidly, because in the pressurized muscle, Ca 2, concentration was probably increased so far as /_z-calpain was activated.
330 In short, at 200MPa, r activated by an increase in Ca ~§ may have decreased thep level of Zz-calpain by autolysis. Suzuki et al. [8] presented direct evidence for the pressure-induced Ca '.+ release from sarcoplasmic reticulum from electron micrographs of the pyroanthimonate-fixed fiber bundles prepared from pressurized muscles. Emori [9] reported that the primary structures of the /.z- and m-calpain large subunits were similar to one another. On the basis of these reports, it was difficult to accept that the two calpains had a different ability to withstand pressurization. From the results,it was concluded that calpain levels remained in muscle pressurized up to 200 MPa, whereas calpastatin level in the muscle was decreased and Ca ~+ concentration increased by the pressurization. Therefore,the total activities of calpains in the pressurized muscle were increased, and could result in tenderization of meat. P?essure Applied
=-Calpain
Control
100
m-Calpain 100
100
IOOMPa
55.9
96.4
39.6
200MPa
5.4
99.5
14.5
300MPa
1.6
8.2
Values are calculated
the on
in the
vitro basis
activities of the
Table 1 Relative levels of calpain and calpastatin after pressurization
Calpastatin
4.3 of each chromatographs.
peak,
4. CONCLUSION From the results, it was concluded that the pressure-induced increase in the amount of protease activity was due to the release of the enzymes from lysosomes. And total activities of calpains in the pressurized muscle were increased by the pressure treatment. These increase of activity may result in tenderization of meat. 5. REFERENCES
1 W. N. Schwartz and J. W. C. Bird, Biochem. J., 167 (1977) 811. 2 A. Okitani, U. Matsukura, H. Kato and M. Fujimaki, J. Biochem., 87 (1980) 1133. 3 D. E. Goll, M. H. Stromer, D. G. Olson, W. R. Dayton, A. Suzuld and R. M. Robson, Proc.of the Meat Industry Res. Conf., Am. Meat Inst. Found. Arlington, Virginia, 1974 4 A. Suzuld and D. E. Goll, Agric. Biol. Chem., 38 (1974) 2167. 5 J. J. Macfarlane, Developments in Meat Science-3(R. A. Lawrie, ed. ), Elsevier Applied Science Publishers, Barking, Essex, 1985 6 E. A. Elgasim and W. H. Kennick, Food Microstructure, 1 (1982) 75. 7 A. Suzuki, K. Kim, N. Homma, Y. Ikeuchi and M. Saito, High Pressure and Biotechnology. (C.Balny et al., eds.), Colloques INSERM/John Libbey Eurotext, Montrouge, 1992 8 Y. Emori, H. Kawasaki, S. Imajoh, S. Kawashima and K. Suzuki, J. Biol. Chem., 261 (1986) 9472. 9 A. Suzuki, A. Okamoto, Y. Ikeuchi and M. Saito, Biosci. Biotech. Biochem., 57 (1993) 862.